Amino-01 (July 2018)

Single Laboratory Validation Report for Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals

Chromatography solvents (mobile phases)

3.4.1 Eluent A (Solvent A) Prepare Eluent A from AccQ•Tag™ Ultra Eluent A concentrate as follows: a. Measure 850 mL of water into a 1-L graduated cylinder. b. In a separate graduated cylinder, measure 150 mL of AccQ•Tag™ Ultra Eluent A concentrate. c. Add the concentrate to the water and mix thoroughly. Note: Eluent A concentrate, once opened, must be stored tightly capped at around 4 °C. Dilute Eluent A is stable for 1 week at room temperature. 3.4.2 Eluent B (Solvent B) AccQ•Tag™ Eluent B is supplied as a working solution; no additional preparation is required. Eluent B, once opened, must be stored tightly capped at around 4 °C for no longer than 1 month. Alternative Eluent B: use HPLC-grade Acetonitrile supplemented with 2 % (W/w) formic acid.

3.4.3 Wash solvents The weak needle wash solvent is 5 % (V/V) acetonitrile in water. The strong needle wash solvent is 95 % (V/V) acetonitrile in water. The seal wash solvent is 50 % (V/V) acetonitrile in water.

Sample analysis

3.5.1 Sample preparation Reconstitute powders by adding 25 g powder to 200 g water and mix thoroughly. Weigh out 220±20 mg reconstituted powders or ready-to-feed liquids into a 10-mL glass tube with screw cap. Report the sample mass to 0.1 mg. Complete to 1100 mg with water. To each tube, add: - 600 μL DDP solution (1% DDP in 0.2 M NaOH) - 600 μL 0.2 M HCl - 200 μL 10 mM Nva stock solution (10 pmol/μL final concentration after derivatization) - 2500 μL phenol/HCl solution (0.1 % phenol in 12 M HCl) Note: phenol/HCl solution has to be added under the hood. Sparge the tube ~5 seconds with a stream of nitrogen to displace oxygen. Close tubes with screw caps and vortex. Note: make sure the caps are perfectly clean (i.e. devoid of any particle) to ensure tightness and avoid evaporation during hydrolysis.

Jaudzems, Guthrie, Lahrichi, and Fuerer – January 2018

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